1. Field of the Invention
The present invention relates to a method for developing a culture medium using genome information and in silico analysis. More particularly, it relates to a method for developing a minimal synthetic medium, the method comprising constructing a metabolic network using genome information of prokaryotic cell or eukaryotic cell, selecting components of the minimal synthetic medium removing any one among external metabolites from the constructed metabolic network and conducting metabolic flux analysis using in silico simulation, and determining a final culture medium by actual culture.
2. Background of the Related Art
The culture of prokaryotic cells or eukaryotic cells in a specific environment requires various nutrients. The specific nutrient requirements vary according to the kind of animals, plants, insects and microorganisms, or the kind of cell tissues or organs to be cultured and thus the medium composition for each culture is different. The culture of eukaryotic cell or prokaryotic cell requires combination medium containing a proper combination of nutrients, hormone, growth factor and blood serum, in general, a medium prepared by adding inorganic salts, amino acids and vitamins to the medium containing the extract of cells or cell lines to be cultured.
In a case of blood serum, although Sato et al established non-blood serum culture method by adding known components to a medium without adding blood serum (Rizzino et al., PNAS, 75:1844, 1978), the final culture medium contains a tissue extract in which various unknown chemicals exist. Some of unknown compounds came from a tissue extract could inhibit the growth of cells and cell lines. Especially, they can cause a fatal risk in patients when cells or cell lines are cultured and used for treatment. For biopharmaceuticals production, they not only decrease the productivity but also increase the costs of separation and purification. Therefore, the development of a culture medium in which all components are known and well clarified is required in order to insure safety in use and elucidate the roles of individual components of culture media for the production of biopharmaceutical products or the treatment through the culture of cell lines or cells of insects, plants and animals.
Complex or semi-defined media which contain peptone, yeast extract, corn steep liquor, blood serum, beef stock, and/or malt extracts, etc, they are composed of numerous unknown compounds, are normally used for the culture of microorganisms. However, their qualities and compositions vary with manufacture dates, manufacturing enterprises, and manufacturing materials. In addition, the prices of them are more expensive than those of chemicals used in preparation of chemically defined, synthetic media. Also, use of a complex medium containing the aforementioned materials cannot guarantee the production of goods with consistent quality through microorganisms cultivation and unknown components in the complex medium cause an increase in the costs of separation and purification. It has been reported that some unknown components present in complex and semi-defined media inhibit cell growth and production of specific metabolites (Zhang et al., Appl. Microbiol. Biotechnol., 51:407, 1997). Moreover, unknown components in the complex medium make it difficult to understand exact cellular metabolic characteristics and thus restrict the development of excellent bacteria using metabolic engineering technique.
Therefore, numerous researches have focused on the development of chemically defined media to overcome shortcomings in using complex media which include the aforementioned materials. In fact, synthetic media have been developed and employed for the production of exopolysaccharides and lactic acid through microbial fermentation (Cocaign-Bousquet et al., J. Appl. Bacteriol., 79:108, 1995; Grobben et al., Appl. Environ. Microbiol., 64:1333, 2003).
To date, a synthetic medium have been developed by using a single omission technique, in which a single component is sequentially eliminated from a culture medium containing all nutrient components. After observing whether cells can grow in a medium in the absence of a single component, components essential for cell growth are identified and a synthetic medium are formulated. (Zhang et al., Appl. Microbiol. Biotechnol., 51:407, 1997). However, a single omission technique is based on a trial and error method, indicating that this method is laborious, time-consuming, and costly. In order to overcome the above shortcomings, although various statistical methods have been developed and employed for identifying components necessary for cell growth and thus formulating a synthetic medium, these methods are also based on a trial and error method and their success is very low.
Various techniques based on statistical method such as Plackett-Burman experimental design, fractional factorial experimental design, central composite experimental design, and response surface technique have been applied to develop a synthetic medium (Dasu et al., J. Microbiol. Biotechnol., 12:355, 2002; Dasu et al., J. Microbiol. Biotechnol., 12:360, 2002; Mantha et al., Bioprocess Eng., 19:285, 1998).
Since human genome map was completed in the 2003 after the completion of human genome project, till now, the whole genome sequences of more than 300 species of organisms has been deciphered. Also, now, many researchers are making a major contribution to reveal genome sequences of various organisms and constructing a metabolic network based on the revealed genome information (Ng et al., PNAS, 97:12176, 2000; Ruepp et al., Nature, 407:466, 2000; Nierman et al., PNAS, 98:4136, 2001; Perna et al., Nature, 409:529, 2001; Adams et al., Science, 287:2185, 2000).
Succinic acid (HOOCCH2CH2COOH) is a high functional basic chemical having various industrial applications ranging from a precursor of chemicals to foods and pharmaceuticals (Zeikus et al., Appl. Microbiol. Biotechnol., 51:545, 1999). Since the usage of succinic acid as a raw material of polybutylene succinate (Ajinomoto, Environ. Rep., 21, 2003) and polyamides, which are major biodegradable polymers (Willke et al., Appl. Microbiol. Biotechnol., 66:131, 2004), is verified recently, its dramatic demand thereof is expected.
Succinic acid can be produced by chemical synthesis and conventional microbial fermentation. Only a small amount of succinic acid is produced through microbial fermentation for specific uses such as foods and pharmaceuticals. On the other hand, most commercial succinic acid is synthesized using liquid propane gas LPG or n-butane from crude oil, and this chemical process yield large amounts of harmful wastes, waste water, and waste gas. Particularly, it has a limitation in that it uses fossil fuel as a starting material, and recent increase of crude oil price causes an increase in the price of succinic acid.
In addition, with an increased interest in eco-friendly processes, many researchers have made extensive efforts to develop economical and eco-friendly succinic acid production process in order to solve the problems caused by the chemical synthetic processes. Recently, the production of succinic acid through microbial fermentation using renewable resources has drawn a significant attention as an alternative succinic acid production process.
Researches on succinic acid production through microbial fermentation can be divided into fermentation process development, the development of separation and purification processes, and the development of cheaper substrates and excellent bacteria. The representative succinic acid-producing bacteria are recombinant E. coli, Anaerobiospirillum and ruminal bacteria Actinobacillus,: Bacteroides, Mannheimia, Succinimonas and Succinivibrio etc.
Chicago University research group in the United States created recombinant E. coli NZN111 strain, in which ldh and pfl genes participating in the production of lactic and formic acids, respectively, were deleted to enhance succinic acid production. This research group further created an AFP111 (ATCC 202021) strain by manipulating a glucose transport gene ptsG in the NZN111 strain (U.S. Pat. No. 5,770,435). Also, the present inventors overexpressed a malic gene (sfcA) involved in the production of succinic acid in the NZN111 strain, which successfully increased succinic acid production as well as effectively prevented pyruvic acid accumulation (Hong et al., Biotechnol. Bioeng., 74:89, 2001). Also, Georgia University research team in the United States made an attempt to increase succinic acid production by an AFP111/pTrc99A-pyc strain obtained by overexpressing pyruvate carboxylase gene(pyc) of Rhizobium etli strain in the AFP111 strain (Vemuri et al., J. Ind. Microbiol. Biotechnol., 28:325, 2001). More recently, Rice University research team in the United States created various recombinant E. coli strains by manipulating genes involved in glycolysis, tricarboxylic acid (TCA) cycle, and glyoxylate pathways, which induced succinic acid production under an aerobic condition (Lin et al., Metabol. Eng., 7:116, 2005; Lin et al., Biotechnol. Bioeng., 90:775, 2005).
Anaerobiospirillum succiniciproducens strain identified in gullet and excrement from German hunting dogs has an ability to produce a large amount of succinic acid under absolute anaerobic conditions (Davis et al., Int. J. Syst. Bacteriol., 26:498, 1976; Samuelov et al., Appl. Environ. Microbiol., 57:3013, 1991). Accordingly, the present inventors have conducted studies with respect to various kinds of carbon sources, nitiride nitrogen sources, and gas components (CO2/H2) for the production of succinic acid using Anaerobiospirillum succiniciproducens. (Lee et al., Enzyme Microbial Technol., 24:549, 1999; Lee et al., Biotechnol. Bioeng., 72:41, 2001). Also, Michigan Biotechnology Institute(MBI) International research group developed a process for the production of succinic acid and purification thereof using the above bacteria (U.S. Pat. No. 5,521,075; U.S. Pat. No. 5,168,055; U.S. Pat. No. 5,143,834).
Among rumen bacteria, Actinobacillus and Menheimia are relatively widely studied. MBI research group created an Actinobacillus succinogenes 130Z strain (ATCC 55618) and other mutants resistant to sodium monofluoroacetate and used them in the succinic acid production process with high concentration (U.S. Pat. No. 5,504,004; U.S. Pat. No. 5,573,931). Recently, there has been an attempt to increase the production of succinic acid through a repeated batch culture by attaching the 130Z strain to a polymer substance (Urbance et al., Appl. Microbiol. Biotechnol., 65:664, 2004). The present inventors isolated Mannheimia succiniciproducens MBEL55E from Korean rumen which has an ability to produce succinic acid with high efficiency (Lee et al., Appl. Microbiol. Biotechnol., 58:663, 2002; KCTC 0769BP, Korean Collection for Type Cultures), and recently announced its whole genome sequences consisting of 2,314,078 base pairs (Hong et al., Nature Biotechnol., 22:1275, 2004).
Especially, the applicants of the present invention created a Mannheimia sp. LPK strain (KCTC 10558BP) in which lactate dehydrogenase (ldhA) and pyruvate formate-lyase (pfl) genes are deleted. Also, the present inventors have created a succinic acid overproducing sp. LPK7 strain obtained by deleting phosphotransacetylase (pta) and acetate kinase (ackA) genes from the LPK strain (WO 05/052135 A1). In the succinic acid production process using the above mentioned bacteria, complex media whose components are chemically undefined, which contains peptone, yeast extracts and/or corn steep liquor, have been used.
As stated the above, the compositions of components present in complex media are not consistent and their prices are expensive than those of chemicals used in the preparation of a synthetic medium. Also, use of the complex medium containing the aforementioned components cannot guarantee the production of goods with consistent quality through the culture of microorganisms and unknown components in the complex medium cause an increase in the costs of separation and purification. It has been known that some of these components inhibit cell growth and the production of specific metabolites. Especially, unknown components in a complex medium make it difficult to elucidate cellular metabolic characteristics and restrict the creation of excellent bacteria through metabolic engineering.
Accordingly, it has been reported that synthetic media of Actinobacillus succinogen known as an important strain producing succinic acid was developed by employing a single omission technique (McKinlay et al., Appl. Environ. Microbiol., 71:6651, 2005). Since the whole genome information of the bacteria cannot be used, the development of synthetic media was achieved by using the previously known information on synthetic culture media of other bacteria. In other words, a vitamin mixture solution containing 10 kinds of vitamins which was used in the culture of methane producing bacteria was used as vitamin components and an amino acid mixture solution containing 12 kinds of amino acids which was used in the culture of Haemophilus influenzae was used as amino acid components. Finally, a synthetic medium containing 3 kinds of amino acids obtained by eliminating each amino acid from the above amino acid mixture solution and the aforementioned 10 kinds of vitamin mixture solution are prepared. However, this method is just one example using the conventional single omission technique. Especially, the culture medium showed cell growth rate of 50% compared with culture media containing 12 kinds of amino acid and 10 kinds of vitamins. Recently, nutrients necessary for the growth of Lactobacillus plantarum are identified using a single omission technique and they are used as basic data for the construction of metabolic network using genome information (Teusink et al., Appl. Environ. Microbiol., 71:7253, 2005). However, attempts on the identification of nutrients necessary for the growth of cells or cell lines from genome information of organisms and the development method of a culture medium using thereof are not achieved yet.
Thus, it is desperately in need to develop a method for preparing efficient culture medium of eukaryotic cell or prokaryotic cell, which is more systematical and accurate, and can reduce research costs so that it can substitute for the conventional single omission technique or statistical method requiring many times, many efforts and a huge amount of research costs. For this, the whole metabolic network based on decoded genome information of organism must be first constructed and based on the constructed metabolic network, nutrients certainly necessary for production of target metabolites and growth of eukaryotic or prokaryotic cells are determined to prepare minimal and optimal synthetic media using the nutrients. Particularly, until now, there has been no attempt to develop a culture medium of eukaryotic cell or prokaryotic cells based on the whole metabolic network using the above described genome information.
Also, amino acids such as alanine, asparagine, glutamic acid, histidine, isoleucine and leucine and vitamins such as ascorbic acid are used widely as a major component of a culture medium, however, the effects thereof on the growth of eukaryotic or prokaryotic cells and the production of target metabolites has not been found yet.
Accordingly, in order to efficient culture medium development for eukaryotic or prokaryotic cells, the present inventors have identified nutrients necessary for the production of succinic acid as a target metabolite and cell growth using metabolic network constructed based on the whole genome information of M. succiniciproducens MBEL55E, and have developed a minimal and optimal synthetic medium whose components are chemically defined and found the function of the culture medium for synthesizing target metabolites. Based on the above finding, the present invention has been completed.